Biology subjects

Leister, D.

Publications and source records attributed to Leister, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Multi-omics dissection of transcriptional and post-transcriptional responses in cyanobacterial high-light adaptation

Understanding how photosynthetic organisms acclimate to excess light is essential for the development of robust chassis for synthetic biology approaches aimed at expanding the photosynthetically active absorption spectrum. High-light (HL) tolerant strains were previously generated by laboratory evolution from a laboratory type (LT) strain of Synechocystis sp. PCC 6803, with tolerance attributed to a small number of specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroduction of these mutations into the LT background was sufficient to confer HL tolerance. However, the mechanisms by which a limited set of point mutations mediates HL tolerance have remained unclear. Here, integrated transcriptomic and proteomic analyses of HL-tolerant strains reveal a coordinated network of responses underlying HL tolerance. The NdhF1F124L mutation increased the accumulation of NDH-1 complex subunits, likely accounting for the previously observed enhancement of cyclic electron flow. In contrast, EF-G2R461C increased the abundance of multiple functional classes of proteins associated with HL tolerance, while post-transcriptionally reducing the level of the phycobilisome linker protein CpcC2, resulting in a decreased antenna size. Integrated analyses further demonstrated that HL tolerance involves transcriptional regulation of protein abundance, including the maintenance of phosphate metabolism. Consistently, overexpression of two genes from the Pho regulon increased HL tolerance. Overall, this study demonstrates how a small number of point mutations in genes with central cellular functions can reprogram the cyanobacterial cell to achieve enhanced tolerance to HL.

evolutionary biology↗

bioRxiv2025

The functional divergence of plant ESCRT components TOL3, SNF7.1, and VPS4 during salt stress response

Salinity, a major abiotic stressor, impacts protein trafficking and alters transport routes. The endosomal sorting complex required for transport (ESCRT)is a key player for membrane protein sorting and is increasingly recognized as pivotal element in abiotic stress responses. However, the functional role of ESCRT proteins in cereal crops under salt stress remains unexplored. Using the as near as possible to nature DRD-BIBLOX (Dark-Root device Brick Black Box), barley (Hordeum vulgare, L.), although relatively salt-tolerant, exhibits impaired germination, seedling growth, and an altered root system architecture (RSA) under high salinity. Shotgun proteomic analysis revealed spatio-temporal regulation of ESCRT proteins in shoots and roots during seedling development. We identified HvSNF7.1 and HvVPS4 as critical, but differently acting modulators of salt stress responses in barley tissues. In this study we further reveal novel functions of ESCRT-0-like TOM-1-like proteins (TOLs): Previously described as gatekeepers of degradative protein sorting and as regulators of abscisic acid signaling, growth, and heat stress responses in Arabidopsis (Arabidopsis thaliana), HvTOLs had not been characterized for their role in salinity adaptation during plant development. Functional analyses demonstrated lethality in Hvtoldouble and Hvtolquadruple mutants under standard growth conditions. In Arabidopsis, loss of AtTOL3 alone impaired germination and root growth under salt stress, underscoring the essential role of individual TOL proteins in the spatio-temporal regulation of abiotic stress adaptation. These findings highlight ESCRT-mediated trafficking as a key determinant of cereal resilience under saline conditions and provide a molecular framework for improving crop stress tolerance.

plant biology↗

bioRxiv2025

Role of Chloroplast Lipid-Remodelling Protein 23 During Cold Acclimation in Arabidopsis thaliana

Cold acclimation is a crucial physiological process that enables plants to adapt to low temperatures. A key aspect of this adaptation is lipid remodeling, which preserves membrane fluidity and integrity under cold stress. Proteins of the chloroplast envelope membranes are increasingly recognized for their role in acclimation to changing environmental conditions. While lipid synthesis occurs at the inner envelope membrane, little is known about specific proteins involved in lipid remodeling during cold acclimation. In this study, we investigate the role of Chloroplast Lipid Remodeling Protein 23 (CLRP23) as a component of the inner chloroplast envelope membrane. Subcellular fractionation combined with protease protection assays provided evidence for its orientation toward the intermembrane space. To explore its function, we analyzed the physiological performance and lipid composition in CLRP23-deficient mutant plants. Under cold stress, we observed significant impairments in photosynthesis and exaggerations in galactolipid response, suggesting CLRP23 is involved in lipid remodeling. Lipid overlay assays, supported by in silico docking analyses, demonstrate that CLRP23 can directly interact with chloroplast lipids, including galactolipids. Complementary transcriptomic and proteomic analyses reveal broader effects on cold-responsive pathways, supporting the view that CLRP23 contributes to the integration of membrane and metabolic responses during acclimation. These findings expand our understanding of protein-mediated processes during cold acclimation.

plant biology↗

bioRxiv2025

High-light adaptation in Synechocystis by accumulating NDH proteins and depleting specific phycobilisome linker proteins

Photosynthetic organisms have evolved mechanisms to manage excess light, crucial for maximizing photosynthetic efficiency. High-light (HL) tolerant Synechocystis sp. PCC6803 strains were developed through laboratory evolution, with tolerance attributed to specific point mutations. Key mutations affected the NDH-1L complex F1-subunit (NdhF1F124L) and translation elongation factor G2 (EF-G2R461C). Reintroducing these mutations into laboratory strains conferred HL tolerance. Comparisons with knockout and overexpressor lines showed NdhF1F124L and EF-G2R461C result in gain of function. Transcriptomic and proteomic analysis unveiled a network of responses contributing to HL tolerance, including maintenance of phosphate metabolism and decreased antenna size by depleting a specific linker protein in EF-G2R461C cells. Consequently, overexpression of Pho regulon genes increased HL tolerance. NdhF1F124L enhances cyclic electron flow (CEF) by increasing NDH-1 complex subunit accumulation. Other HL-adapted strains demonstrated that increased CEF and decreased antenna size are recurring outcomes, achievable through various mutations. This study demonstrates how limited mutations can reconfigure cells for enhanced HL tolerance, offering insights for improving photosynthetic efficiency.

physiology↗

bioRxiv2025

Improving tolerance to fluctuating light through adaptive laboratory evolution in the cyanobacterium Synechocystis

Fluctuating light (FL) poses a serious challenge to photosynthetic organisms like cyanobacteria, disrupting carbon assimilation and damaging photosystems. While key components of cyanobacterial FL tolerance have been identified, their genetic enhancement remains unexplored. We applied adaptive laboratory evolution to Synechocystis PCC 6803 under two complex FL regimes, including one lethal to the starter strain (LT), to generate FL-adaptive alleles. Our analysis revealed 44 fully segregated novel mutations in 24 monoclonal evolved strains, 28 of which affected proteins or structural RNAs. We focused on three mutations for further study. Mutations in Pam68, involved in photosystem II (PSII) assembly, and Sll0518 were present in all evolved strains, indicating early emergence. These mutations increased tolerance to non-lethal FL conditions when introduced into LT, with the Pam68 mutation possibly protecting PSI by increasing the proportion of less active PSII monomers. A gain-of-function mutation in RpaB, regulator of phycobilisome association B, was found in three strains tolerant to lethal FL. When introduced into LT, this mutation significantly increased tolerance to both lethal FL and high light conditions, associated with downregulation of photosystem accumulation and light harvesting. As RpaB has plant homologs, this finding could potentially be used to improve agricultural productivity under variable light conditions.

evolutionary biology↗

bioRxiv2024

Regulation of plant metabolism under elevated CO2

Plant responses to changing environments afford complex regulation at transcriptome and proteome level to maintain metabolic homeostasis. Homeostasis itself constitutes a complex and dynamic equilibrium of metabolic reactions and transport processes among cellular compartments. In the present study, we aimed at the highest possible resolution of this network by combining analysis of transcriptome, proteome and subcellular resolved metabolome of plants exposed to rising carbon dioxide concentrations over a time course of one week. To prove suitability of our approach, we included mutants affected in photorespiratory metabolism and, thus, should deviate from the wildtype in their response to elevated CO2. Our multi-omics analysis revealed that the hpr1-1 mutant, defective in peroxisomal hydroxypyruvate reduction, is also affected in cytosolic pyruvate metabolism, reaching out to cysteine synthesis, while the hexokinase mutant hxk1 displays a disturbed redox balance upon changing CO2 levels. For the third mutant, defective in the mitochondrial protein BOU, we found compelling evidence that the function of this transporter is related to lipoic acid metabolism, thus challenging current interpretations. This demonstrates that the combined omics approach introduced here opens new insights into complex metabolic interaction of pathways shared among different cellular compartments.

plant biology↗

bioRxiv2024

Flavonoid metabolism and its different physiological roles in plant cold acclimation

Flavonoids represent a diverse group of plant secondary metabolites which are also discussed in context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non-aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal subcellular regulation during cold acclimation in two flavonoid-deficient mutants. During the first three days of a two-week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected which had significant effects on regulation of the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation which resulted in a significant decrease of total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment.

plant biology↗

bioRxiv2024

Arabidopsis GENOMES UNCOUPLED PROTEIN1 binds to plastid RNAs and promotes their maturation

Plastid biogenesis and the coordination of plastid and nuclear genome expression through anterograde and retrograde signaling are essential for plant development. GENOMES UNCOUPLED1 (GUN1) plays a central role in retrograde signaling during early plant development. The putative function of GUN1 has been extensively studied, but its molecular function remains controversial. Here, we evaluate published transcriptome data and generate our own data from gun1 mutants grown under signaling relevant conditions to show that editing and splicing are not relevant for GUN1-dependent retrograde signaling. Our study of the plastid (post)-transcriptome of gun1 seedlings with white and pale cotyledons demonstrates that GUN1 deficiency significantly alters the entire plastid transcriptome. By combining this result with a PPR code-based prediction and experimental validation by RNA immunoprecipitation experiments, several targets of GUN1 were identified, including 23S rRNA, tRNAs and RNAs derived from ycf1.2 and the ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndhD gene cluster. The absence of plastid rRNAs and the significant reduction of almost all plastid transcripts in white gun1 mutants account for the cotyledon phenotype. Our study identifies RNA binding and maturation as the long-sought molecular function of GUN1 and resolves long-standing controversies. We anticipate that our findings will serve as a basis for subsequent studies investigating the mechanism of plastid gene expression and will facilitate the elucidation of GUN1s function in retrograde signaling.

plant biology↗

bioRxiv2023

A pgr5 suppressor screen uncovers a distinct mechanism safeguarding the cytochrome b6f complex from damage through PGR5

PROTON GRADIENT REGULATION5 (PGR5) is thought to promote cyclic electron flow (CEF) and its deficiency causes increased photosensitivity of photosystem I (PSI), leading to lethality under fluctuating light (FL). By screening for suppressor mutations that rescue FL lethality of pgr5 plants, we identified a portfolio of mutations affecting 12 photosynthesis-related proteins. Six are required for proper PSII function, one (CcdA) promotes cytochrome (cyt) b6f assembly, and another (PAA1) provides plastocyanin with its copper cofactor. Two other mutations are associated with the chloroplast FBPase cFBP1. This, together with targeted knockout of other genes in the pgr5 background, suggests three pathways to restore FL viability: (i) reduced electron flow to PSI due to defects in PSII, cyt b6f or plastocyanin but not PSI, (ii) increased electron flow from PSI due to inactivation of ACHT2, a regulator of cFBP1 activity, and (iii) hyperactivity of the NDH-dependent CEF due to inactivation of cFBP1. The remaining two suppressor mutations affected the cyt b6f complex. PFSC1 controls cyt b6f accumulation at early developmental stages. DEIP1/NTA1, previously suggested to be essential for cyt b6f assembly, appears to protect cyt b6f from deleterious effects of PGR5, since plants lacking both DEIP1/NTA1 and PGR5 are viable and accumulate cyt b6f.

plant biology↗

bioRxiv2023

Identification of a highly drought-resistant pp7l hda6 mutant

Plants have evolved efficient strategies to cope with drought stress, including stomata closure, significant changes in nuclear gene expression, and epigenetic mechanisms. Previously, we identified Arabidopsis thaliana PROTEIN PHOSPHATASE7-LIKE (PP7L) as an extrachloroplastic protein that promotes chloroplast development and high light, and salt tolerance. Here, we demonstrate that the pp7l mutant can withstand prolonged periods of drought stress. Interestingly, chloroplast development in pp7l recovers under drought conditions, despite growth of the mutant is impaired under normal growth conditions. To assess the (post)transcriptional changes occurring in the pp7l mutant under different durations of drought exposure, we used long non-coding RNA-sequencing. Compared to the previously reported drought-responsive changes in the wild type, the drought-responsive changes detected in the pp7l mutant were negligible. Our analysis of data generated in this study and previously motivated us to create a pp7l hda6 mutant, which exhibits remarkable drought resistance. Notably, the growth penalty associated with pp7l was alleviated in the double mutant, ruling out a dwarf effect on the drought-tolerant trait of this genotype.

plant biology↗

bioRxiv2023

UPP affects chloroplast development by interfering with chloroplast proteostasis

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme which appears to have a previously unknown, moonlighting activity. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial for chloroplast development and growth. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis suggest that UPP plays a role in chloroplast proteostasis, especially under high light (HL). Immunoblots of mature plants and a de-etiolation experiment with young seedlings revealed PetC, the iron-sulfur protein of the cytochrome b6f complex, as a putative UPP target. In addition, UPP and PetC were identified in a high molecular weight complex. Consistently, we show that PetC is massively reduced in UPP knock-down plants. The block observed in photosynthetic electron transport, as evidenced by reduced high light-induced non-photochemical quenching (NPQ) but increased unregulated energy dissipation (NO), might be therefore a consequence of reduced PetC. After HL treatment, UPP amiRNA mutants showed impaired photosynthesis and reduced carbohydrate contents, resulting in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline and fumarate were found to be reduced. Proteases, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery, were up-regulated. In sum, our work suggests that UPP assists in stabilizing and protection of PetC during the assembly of the Fe-S cluster and targeting to the thylakoid.

molecular biology↗

bioRxiv2023

Spliceosomal complex components are critical for adjusting the C:N balance during high-light acclimation

Plant acclimation to an ever-changing environment is decisive for growth, reproduction and survival. Light availability limits biomass production on both ends of the intensity spectrum. Therefore, the adjustment of plant metabolism is central to high-light (HL) acclimation, and accumulation of photoprotective anthocyanins is commonly observed. However, mechanisms and factors regulating the HL acclimation response are less clear. Two Arabidopsis mutants of spliceosome components exhibiting a pronounced anthocyanin overaccumulation in HL were isolated from a forward genetic screen for new factors crucial for plant acclimation. Time-resolved physiological, transcriptome and metabolome analysis revealed a vital function of the spliceosome components for rapidly adjusting gene expression and metabolism. Deficiency of INCREASED LEVEL OF POLYPLOIDY1 (ILP1), NTC-RELATED PROTEIN1 (NTR1), and PLEIOTROPIC REGULATORY LOCUS1 (PRL1) resulted in a marked overaccumulation of carbohydrates and strongly diminished amino acid biosynthesis in HL. While not generally limited in N-assimilation, ilp1, ntr1, and prl1 mutants showed higher glutamate levels and reduced amino acid biosynthesis in HL. The comprehensive analysis reveals a function of the spliceosome in the conditional regulation of the carbon:nitrogen-balance and the accumulation of anthocyanins during HL acclimation. The importance of gene expression, metabolic regulation, and re-direction of carbon towards anthocyanin biosynthesis for HL acclimation are discussed.

molecular biology↗

bioRxiv2023

Probing the physiological role of the plastid outer-envelope membrane using the oemiR plasmid collection

Plastids are the site of complex biochemical pathways, most prominently photosynthesis. The organelle evolved through endosymbiosis with a cyanobacterium, which is exemplified by the outer envelope (OE) membrane that harbors more than 40 proteins in Arabidopsis. Their evolutionary conservation indicates high significance for plant cell function. While a few proteins are well-studied as part of the protein translocon complex the majority of OE protein (OEP) functions is unclear. Gaining a deeper functional understanding has been complicated by the lack of observable loss-of-function mutant phenotypes, which is often rooted in functional genetic redundancy. Therefore, we designed OE-specific artificial micro RNAs (oemiRs) capable of downregulating transcripts from several loci simultaneously. We successfully tested oemiR function by performing a proof-of-concept screen for pale and cold-sensitive mutants. An in-depth analysis of pale mutant alleles deficient in the translocon component TOC75 using proteomics provided new insights into putative compensatory import pathways. The cold stress screen not only recapitulated three previously known phenotypes of cold-sensitive mutants, but also identified four mutants of additional oemiR OE loci. Altogether our study revealed a role of the OE to tolerate cold conditions and showcasts the power of the oemiR collection to research the significance of OEPs.

plant biology↗

bioRxiv2023

Arabidopsis thaliana BBX14 is a target of GLK1 and involved in high-light acclimation, photomorphogenesis and GUN-type retrograde signaling

Development of photosynthetically competent seedlings requires both light and retrograde biogenic signaling pathways. The transcription factor GLK1 functions at the interface between these pathways, and receives input from the biogenic-signaling integrator GUN1. BBX14 was previously identified, together with GLK1, in a core module that mediates the response to high light levels and biogenic signaling. To gain insight into the function of BBX14, we generated BBX14 overexpressors and CRISPR/Cas-mediated bbx14 mutant plants, conducted high-light, RT-qPCR and ChIP-Seq experiments, measured photosynthetic parameters, chlorophyll contents and growth rates, and analyzed alterations in transcriptomics. We found that, although overexpression of BBX14 is deleterious under normal growth conditions, BBX14 is needed to acclimate plants to high light stress. BBX14 is a direct target of GLK1, and RNA-Seq analysis suggests that BBX14 is involved in the circadian clock. Knockout of BBX14 results in a long-hypocotyl phenotype that depends on a retrograde signal, and BBX14 expression during biogenic signaling requires GUN1. Finally, we clarify the role of BBX14 in GUN-type biogenic signaling. We conclude that BBX14 is an integrator of photomorphogenetic and biogenic signals, and suggest that BBX14 is a nuclear target of retrograde signals downstream of the GUN1/GLK1 module.

plant biology↗

bioRxiv2023

A bench-top dark-root device built with LEGO bricks enables a non-invasive plant root development analysis in soil conditions mirroring nature

Roots are the hidden parts of plants, anchoring their above ground counterparts in the soil. They are responsible for water and nutrient uptake, as well as for interacting with biotic and abiotic factors in the soil. The root system architecture (RSA) and its plasticity are crucial for resource acquisition and consequently correlate with plant performance, while being highly dependent on the surrounding environment, such as soil properties and therefore environmental conditions. Thus, especially for crop plants and regarding agricultural challenges, it is essential to perform molecular and phenotypic analyses of the root system under conditions as near as possible to nature (#asnearaspossibletonature). To prevent root illumination during experimental procedures, which would heavily affect root development, dark-root (D-Root) devices (DRDs) have been developed. In this article, we describe the construction and different applications of a sustainable, affordable, flexible, and easy to assemble open-hardware bench-top LEGO(R) DRD, the DRD-BIBLOX (Brick Black Box). The DRD-BIBLOX consists of one or more 3D-printed rhizoboxes which can be filled with soil, while still providing root visibility. The rhizoboxes sit in a scaffold of secondhand LEGO(R) bricks, which allows root development in the dark as well as non-invasive root-tracking with an infrared (IR) camera and an IR light emitting diode (LED) cluster. Proteomic analyses confirmed significant effects of root illumination on barley root and shoot proteome. Additionally, we confirmed the significant effect of root illumination on barley root and shoot phenotypes. Our data therefore reinforces the importance of the application of field conditions in the lab and the value of our novel device, the DRD-BIBLOX. We further provide a DRD-BIBLOX application spectrum, spanning from investigating a variety of plant species and soil conditions as well as simulating different environmental conditions and stresses, to proteomic and phenotypic analyses, including early root tracking in the dark.

plant biology↗

bioRxiv2022

Response of the organellar and nuclear (post)transcriptomes of Arabidopsis to drought stress

Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used long non-coding RNA-sequencing to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0 and a mutant (protein phosphatase 7-like, pp7l), from which we demonstrated that it can survive long periods of drought stress. The changes detected in the pp7l mutant were marginal, while in the wild type chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Also, transcriptional activation of transposable elements played only a minor role. Conversely, alternative splicing (AS) affected nearly 2,000 genes (11% of expressed nuclear genes). Of these, 25% underwent isoform switching, and 15% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought. Moreover, altering the relative contributions of spliced isoforms might enhance drought resistance. For instance, our data imply that accumulation of a non-functional FLM (FLOWERING LOCUS M) isoform - and not the ratio of functional isoforms as suggested for temperature responses - accounts for the early-flowering phenotype under drought conditions.

plant biology↗

bioRxiv2021

AtCGL160 recruits chloroplast coupling factor 1

ATP synthases couple the generation of chemical energy to a transmembrane electro-chemical potential. Like ATP synthases in bacteria and mitochondria, chloroplast ATP synthases consist of a membrane-spanning (CFO) and a soluble coupling factor (CF1). Accessory factors facilitate subunit production and orchestrate the assembly of the functional CF1-CFO complex. It was previously shown that the accessory factor CGL160 promotes the formation of plant CFO and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. In this study, we show that the N-terminal portion of CGL160 (AtCGL160N), which is specific to the green lineage, is required for late steps in CF1-CFO assembly in Arabidopsis thaliana. In plants that lacked this stroma-exposed domain, photosynthesis was impaired, and amounts of CF1-CFO were reduced to about 65% of the wild-type level. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of CF1 sub-complexes in the stroma relative to the wild type and the CF1 assembly mutant atcgld11-1. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF1 subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the thylakoid-proximal domain of CF1-{beta} that includes the conserved DELSEED motif. We therefore propose that AtCGL160 has acquired an additional function in the recruitment of soluble CF1 to a membrane-integral CFO sub-complex, which is critical for the modulation of CF1-CFO activity and photosynthesis in chloroplasts.

plant biology↗

bioRxiv2021

The RNA-binding protein RBP45D of Arabidopis plays a role in epigenetic control of flowering time and DCL3-independent RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) helps to defend plants against invasive nucleic acids. In the canonical form of RdDM, 24-nt small interfering RNAs (siRNAs) are produced by DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system, in which transcriptional gene silencing (TGS) is independent of DLC3. A forward genetics screen performed with this system identified both known components of RdDM, and the RNA-binding protein RBP45D. RBP45D promotes DNA methylation, and its loss delays flowering, especially at high temperature, presumably mediated by elevated FLC levels. RBP45D is localized to the nucleus, where it is associated with snRNAs and snoRNAs. RBP45D maintains siRNA production originating from the LUC transgene, but does not alter mRNA levels or affect processing of transcripts of known RdDM genes. We suggest that RBPD45 facilitates DCL3-independent siRNA production by stabilising either the precursor RNA or the - as yet unidentified - slicer protein.

plant biology↗